RU2428182C1 - Antimicrobial substance - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to synthetic biologically active substances of heterocyclic series, possessing antimicrobial activity and represents products of modification of known β-lactam antibiotic - ampicillin, including all spatial isomers of claimed substance, all their tautomeric and salt forms. Antomicrobial substance with general formula:

, where R is selected from group: H, methyl, ethyl, n-alkyl (C3-C9), phenyl; X1 and X3 are selected from group: CH₂, O or N (NH or NR, where R - methyl, ethyl, linear or branched alkyl, alkenyl or cycloalkyl C3-C10, CH₂CH₂OEt, CH₂CH₂CH₂OMe, Ph, benzole derivative, substituted one or several times with linear or branched alkyl, alkenyl or cycloalkyl C1-C9, halogen (F, O or Br), CF₃, methoxy group or other alkoxy group, carboxyl or carboxyalkyl, or simultaneously by several of said substituents, CH₂Ph, CH₂CH₂Ph, CH(CH₃)Ph, CH₂C₆H₄(OMe-p), CH₂C₆H₄(F-p), CH₂CH₂C₆H₄(F-p), CH₂CH₂C₆H₃(3,4-diOMe); 2-furylmethyl, 2-(2-thienyl)ethyl; X2 is selected from group: C=0, C=S, CH₂, CMe₂, C(C₅H₁₀-cyclo).

EFFECT: invention provides biologically active substance with high antibacterial activity and wide spectrum of action, efficiently influencing bacteria, present in human cells and in composition of biofilms.

11 tbl, 12 ex

Description

The invention relates to synthetic biologically active substances of a heterocyclic series having antimicrobial activity, and is a modification of the well-known β-lactam antibiotic - ampicillin, including all spatial isomers of the claimed substance, all their tautomeric and salt forms; The invention can be used in the treatment of diseases caused by infections, as well as for similar purposes in veterinary medicine.

Penicillin antibiotics are of great importance in the treatment of bacterial infections, continuing to be one of the most important and widely used groups of antimicrobials. The appearance of penicillin in medical practice in 1940 was an outstanding achievement, which made it possible to effectively treat many dangerous infections. In the future, many other penicillin preparations were created, which greatly expanded the possibilities of treating infectious diseases [Mashkovsky M.D. Medicines - 15th ed., Rev., Rev. and additional - M.: New Wave, 2006. - 1206 p.]. However, the search for new, more effective antibiotics continues today. The need to create new antibiotics is associated with the emergence of microbial strains that are resistant to existing drugs, and the discovery of the possibility of microbial life in communities called “biofilms”. Bacterial biofilms represent the main form of existence of microbes in vivo, including in animals and humans. In biofilms, bacteria and fungi are protected from environmental factors by additional membranes that prevent antibiotics from reaching microbial cells (Davies D. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2003; 2: 114-122; Campanac C., Pineau L., Payard A., Baziard-Mouysset G., Roques C. Interactions between Biocide Cationic Agents and Bacterial Biofilms. Antimicrob Agents Chemother 2002; 46: 1469-1474; Tetz VV, Korobov VP, Artemenko NK, Lemkina LM, Panjkova NV, Tetz GV Extracellular phospholipids of isolated bacterial communities Biofilms 2004; 1: 149-155; BBTets, A.L. Bobrov, L.Yu. Orekhova, D.V. Levkovich, D.S. Shcherbakova, G.V. Tets, A. A. Domorad.).

Known antibiotics of the penicillin series, including its semisynthetic derivatives, penetrate poorly into human cells and bacterial biofilms, as a result, have a weak effect on the bacteria that are in them, which accelerates the variability of the bacteria that form them and, as has recently been shown, increase the risk of chronic infections (Anderl, JN, Franklin MJ, Stewart PS Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 2000.44: 1818-1824. Zahller J., Stewart PS Transmission Electron Microscopic Study of Antibi Action on Klebsiella pneumoniae Biofilm Antim icrob Agents Chemother. 2002; 46: 2679-2683).

The closest chemical structure and purpose to the claimed antimicrobial substance is the antibiotic ampicillin, which was chosen as a prototype, see Mashkovsky M.D. Medicines Medicine, part 2.- M., 1985, pp. 212-215.

Ampicillin has a wide spectrum of antibacterial activity, has low toxicity.

The pharmacokinetics of ampicillin allows the use of the drug to treat infections of various localization.

However, ampicillin does not affect bacteria in human and animal cells, and weakly affects bacteria that are in bacterial biofilms.

The objective of the invention is to create a biologically active substance with high antibacterial activity and a wide spectrum of action, effectively acting on bacteria that are in human cells and in biofilms.

The problem is solved by the synthesis of antimicrobial substances of the general formula (I):

where R is selected from the group: H, methyl, ethyl, n-alkyl (C3-C9), phenyl;

X1 and X3 are selected from the group: CH ₂ , O or N (NH or NR, where R is methyl, ethyl, linear or branched alkyl, alkenyl or cycloalkyl C3-C10, CH ₂ CH ₂ OEt, CH ₂ CH ₂ CH ₂ OMe , Ph, a benzene derivative substituted one or more times by linear or branched alkyl, alkenyl or cycloalkyl C1-C9, halogen (F, C1 or Br), CF ₃ , methoxy or another alkoxy, carboxyl or carboxyalkyl, or several of the following substituents, CH ₂ Ph, CH ₂ CH ₂ Ph, CH (CH ₃ ) Ph, CH ₂ C ₆ H ₄ (OMe-p), CH ₂ C ₆ H ₄ (Fp), CH ₂ CH ₂ C ₆ H ₄ (Fp) , CH ₂ CH ₂ C ₆ H ₃ (3,4-diOMe); 2-furylmethyl, 2- (2-thienyl) ethyl;

X2 is selected from the group: C = O, C = S, CH ₂ , CMe ₂ , C (C ₅ H ₁₀ cyclo).

The invention extends to all spatial isomers of the claimed substances, all their tautomeric and salt forms.

The inventive substances of General formula (I) can be used in the form of neutral forms or in the form of salts with cations of sodium, potassium, lithium, ammonium, magnesium, calcium or other pharmacologically acceptable cations.

Table 1 The structure of the most active claimed derivatives of ampicillin of General formula I Substance number R X1 X2 X3 1a H NH C = o NH 1b H NH C = o NMe 1c H NMe C = o NMe 1g H N-n-neh C = o NH 1d H Nch ₂ ph C = o NH 1st H NCH ₂ C ₆ H ₄ OMe-p C = o NH 1g H NMe C = s NMe 1z H NCH ₂ CH = CH ₂ C = s NH 1i H N-Cyclopropyl C = s NH 1 to H NC ₆ H ₅ C = s NH 1l H N-o-Tolil C = s N-o-Tolil 1m H ABOUT CMe ₂ O 1n H CH ₂ CH ₂ CH ₂ 1o H N-CH ₂ CH ₂ OEt C = s NH 1p H N-Cyclohexyl C = s NH 1p H NC ₆ H ₄ OMe-p C = s NH 1s Me NMe C = o NMe

The applicant has not identified sources containing information on technical solutions identical to the present invention, which allows us to conclude that it meets the criterion of "Novelty."

Biological activity is found to one degree or another in all representatives of this group with R, X1, X2, X3 listed in the general formula (I). The method of synthesis of the claimed substance is also common to all members of the group. Thus, not the specific structure of the radicals and substituents R, X1, X2, X3, which affects only the quantitative level of biological activity, but the belonging of these radicals to the chemical groups listed in general formula (I) is of fundamental importance.

The applicant has not identified sources that would contain information about the influence of the distinguishing features of the invention on the achieved technical result. The specified new property of the object determines, according to the applicant, the invention meets the criterion of "Inventive step".

The inventive substances are synthesized in accordance with Scheme 1. The interpretation of the radicals in the structure of the intermediate reagents 2, 3 and 4 is shown under Scheme 1, and in the target substances 1 in Table 1.

Scheme 1

R1 = H (1a-p, 3a-p), R1 = Me (1c, 3c)

X1 = X3 = H, X2 = CO (1a, 2a, 3a); X1 = NMe, X2 = CO, X3 = NH (1b, 2b, 3b); X1 = X3 = NMe, X2 = CO (1c, s, 2c, s, 3c, s); X1 = Nn-Hex, X2 = CO, X3 = NH (1g, 2g, 3g); X1 = NCH ₂ Ph, X2 = CO, X3 = NH (1d, 2d, 3d); X1 = NCH ₂ C ₆ H ₄ OMe-p, X2 = CO, X3 = NH (1e, 2e, 3e); X1 = X3 = NMe, X2 = CS (1g, 2g, 3zh); X1 = NCH ₂ CH = CH ₂ , X2 = CS, X3 = NH (1s, 2s, 3s); X1 = N-Cyclopropyl, X2 = CS, X3 = NH (1i, 2i, 3i); X1 = NC ₆ H ₅ , X2 = CS, X3 = NH (1k, 2k, 3k); X1-X3 = N-o-Tolyl, X2 = CS (1l, 2l, 3l); X1 = X3 = O, X2 = CMe ₂ (1 m, 2 m, 3 m); X1 = X2 = X3 = CH ₂ (1n, 2n, 3n); X1 = N-CH ₂ CH ₂ OEt, X2 = CS, X3 = NH (1 °, 2 °, 3 °); X1 = N-Cyclohexyl, X2 = CS, X3 = NH (1p, 2p, 3p); X1 = NC ₆ H ₄ OMe-p, X2 = CS, X3 = NH (1p, 2p, 3p).

The synthesis of the claimed substance for all members of the group consists of two main stages.

1) Obtain β-tricarbonyl reagent (3) from the corresponding cyclic β-dicarbonyl reagent (2) (a derivative of barbituric, 2-thiobarbituric acid, Meldrum acid or cyclohexane-1,3-dione) by acylation (formylation) with the corresponding acylating (formylating) agent. The resulting reagent 3 is purified or used in its raw form (without purification).

2) Get the target compound (1a-c) from the reagent 3 synthesized in the first stage and ampicillin (4).

Synthesis can be carried out in one or two stages. The synthesis process of target compounds 1a-c is considered to be one-step, if intermediate reagent 3 is used without purification, and if reagent 3 is isolated in pure form, then the process is considered to be two-step.

The invention is illustrated by the following examples of the synthesis of intermediate substances, examples of the synthesis of the claimed substance, tables of yields and characteristics of the target substances and tables of experimental results to determine the biological properties of the claimed substances, where:

examples 1 - a specific embodiment of the 1st stage of the synthesis of the claimed substance (obtaining an intermediate reagent 3);

examples 3 and 4 are specific embodiments of the 2nd stage of the synthesis of the claimed substance (obtaining target substances 1a-c);

experiment 1 - determination of antibacterial activity;

experiment 2 - determination of the activity of the claimed substances against resistant bacterial strains;

experiment 3 - determination of acute toxicity;

table 1 - structure of the most active representatives of the claimed substances 1a-c;

table 2 - source reagents and the yield of target substances 1a-c;

table 3 - data of spectra of ¹ H NMR of the target substances 1a-c;

table 4 - data of elemental analysis of the target substances 1a-c;

table 5 - the results of determining the antibacterial activity of the claimed substances in vitro in comparison with standards;

table 6 - the results of determining the activity of the claimed substances against resistant bacterial strains;

table 7 - the results of the determination of acute toxicity of the claimed substances.

Example 1. An embodiment of the 1st stage of the synthesis of the claimed substance (obtaining an intermediate β-tricarbonyl reagent 5-formylbarbituric acid (3A)).

Scheme 2

6.4 g (0.05 mol) of barbituric acid 2a, 20 ml of dimethylformamide and 7.4 g (0.05 mol) of triethyl ether of orthoformic acid 5 are charged into a 100 ml flat-bottomed flask equipped with a reflux condenser. The flask is heated to 120 ° C with gradual distillation of the condensate and incubated at this temperature for 15 minutes Then the reaction mixture was cooled to room temperature, 50 ml of ether was added and stirred. The precipitate was filtered off, washed with ether and dried. 6.1 g of product 3a are obtained in the form of light yellow crystals with a _mp > 250 ° C (decomp.).

Example 2. An embodiment of the 1st stage of the synthesis of the claimed substance (obtaining an intermediate β-tricarbonyl reagent - 1,3-dimethyl-5-formylbarbituric acid (3B)).

Scheme 3

6.24 g (0.04 mol) of 1,3-dimethyl barbituric acid 2c and 7.4 g (0.05 mol) of triethyl ether of orthoformic acid 5 are charged into a 100 ml flat-bottomed flask equipped with a reflux condenser. The flask is placed in an oil bath at 70 ° C and in the bath temperature is adjusted to 120 ° C for 10 min with a gradual distillation of the condensate released. Then the reaction mixture was cooled to 60 ° C and washed with hot hexane. The hexane extract was drained from the precipitate, and the precipitate was dissolved without heating in 40 ml of an aqueous solution of 1.6 g (0.04 mol) of NaOH. The resulting solution was filtered through a paper filter, the filtrate was cooled to 10 ° C and acidified with concentrated hydrochloric acid to pH 1. The precipitate formed was washed with cold water and dried in a vacuum desiccator over P ₂ O ₅ . 6.6 g of product 3c are obtained in the form of light yellow crystals with a _{mp of} 111 ° C.

In a similar manner, other β-tricarbonyl reagents 3b, gc are obtained from the corresponding cyclic β-dicarbonyl compounds 2.

Example 3. An embodiment of the 2nd stage of the synthesis of the claimed substance (obtaining the target substance is 3,3-dimethyl-7-oxo-6 - [(2 - {[(2,4,6-trioxotetrahydropyrimidine-5 (2H) -ilidene ) phenyl] amino} propanoyl) amino] -4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid (1a)).

Scheme 4

In a flask with a capacity of 150 ml, 0.01 mol (3.49 g) of ampicillin 4 (or an equivalent amount of ampicillin sodium salt) is dissolved in 50 ml of water with heating. A hot solution of 0.01 mol (1.84 g) of 5-formyl barbituric acid 3a and 0.02 mol (1.64 g) of sodium acetate in 30 ml of water is added to this solution. The resulting reaction mixture was heated for 30 min at 70 ° C, then cooled to room temperature and acidified with hydrochloric acid to pH 1-2. The precipitate formed is filtered, washed with water, then with aqueous alcohol and dried in a vacuum desiccator over P ₂ O ₅ . 4.12 g of product 1a are obtained in the form of a pale yellow crystalline powder. Yield 80%.

Example 4. An embodiment of the 2nd stage of the synthesis of the claimed substance is to obtain the target substance - 3,3-dimethyl-7-oxo-6 - [(2 - {[(1,3-dimethyl-4,6-dioxo-2- thioxotetrahydropyrimidin-5 (2H) -ylidene) phenyl] amino} propanoyl) amino] -4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid (1h).

Scheme 5

A 100 ml flask equipped with a reflux condenser was charged with 6.24 g (0.04 mol) of 1,3-dimethyl-2-thiobarbituric acid 2g and 7.4 g (0.05 mol) of orthoformic acid triethyl ester. The flask was placed in an oil bath at 70 ° C and the temperature of the bath was brought to 110 ° C over 10 minutes with a gradual distillation of the condensate. Then the reaction mixture was cooled to room temperature, 60 ml of an aqueous solution of 0.1 mol of sodium acetate was added to it, and stirred at 30 ° C for 10 min. The undissolved residue is filtered off, the filtrate is transferred to a 200 ml flask and 0.04 mol of ampicillin 4 is added. The reaction mixture is heated at 70 ° C for 30 minutes, then filtered, the filtrate is cooled to room temperature and acidified with hydrochloric acid to pH 1-2. The precipitate formed is filtered, washed with water, then with aqueous alcohol and dried in a vacuum desiccator over P ₂ O ₅ . 13.43 g of product 1g are obtained in the form of a crystalline cream-colored powder.

Example 5. An embodiment of the 2nd stage of the synthesis of the claimed substance 1 (General synthesis method, see Scheme 1).

In a flask with a capacity of 150 ml, 0.01 mol of ampicillin 4 (or an equivalent amount of ampicillin sodium salt) is dissolved in 50 ml of water with heating. A hot solution of 0.01 mol β-tricarbonyl reagent 0.02 mol (1.64 g) sodium acetate in 30 ml of water is poured into this solution. The resulting reaction mixture was heated for 30 min at 70 ° C, then cooled to room temperature and acidified with hydrochloric acid to pH 1-2. The precipitate formed is filtered, washed with water, then with aqueous alcohol and dried in a vacuum desiccator over P ₂ O ₅ . The yield of the target product 1a-c is shown in table 2, the spectral characteristics and data of elemental analysis in tables 3 and 4.

table 2 The starting reagents for the synthesis and yield of target substances Target Substance Number Starting β-tricarbonyl reagent Synthesis Method (Example No.) The yield of the target substance,% 1a 3a 3 78 1a 3a 5 78 1b 3b 5 76 1c 3c four 71 1c 3c 5 82 1g 3G 5 79 1d 3d 5 85 1st 3rd 5 88 1g 3g 5 83 1z 3z 5 80 1i 3rd and 5 80 1 to 3k 5 80 1l 3l 5 69 1m 3m 5 64 1n 3n 5 67 1o 3o 5 85 1p 3p 5 82 1p 3p 5 82 1s 3s 5 45

Table 3 Spectra data of ¹ H of the target substances in DMSO-d ₆ No. of matter
wa Chemical shift, in ppm CMe ₂ , s + s CHCOO with ∗ CH-CH, 2H, m CHP h d ∗ Arh m = CH, d NHCO s ∗ NHC = d ∗ Other h 1a 1.43 + 1.50 4.13 5.45 5.61 7.33 8.02 9.35 10.45 10.61 + 10.74 (2H, s + s, NH) 1b 1.40 + 1.52 4.13 5.41 5.63 7.30 8.08 9.39 10.50 3.12 (3H, s, NMe), 10.72 (1H, s, NH) 1c 1.33 + 1.45 4.10 5.34 5.49 7.22 8.15 8.74 10.55 3.08 + 3.19 (6H, s + s, NMe) 1g 1.42 + 1.47 4.11 5.40 5.63 7.30 8.08 9.39 10.50 0.92 (3H, t, Me), 1.20-1.42 (8H, m, CH ₂ ), 3.62 (2H, q, NCH ₂ ), 10.66 (1H, s, NH) 1d 1.45 + 1.53 4.16 5.41 5.61 7.23 10H 8.15 9.27 10.85 4.93 (2H, d, ab-system, NCH ₂ ), 10.68 (1H, s, NH) 1st 1.45 + 1.53 4.16 5.41 5.60

Continuation of table 3 1g 1.36 + 1.44 4.11 5.35 5.47 7.25 8.10 8.53 11.26 3.52 + 3.58 (6H, s + s, NMe) 1z 1.43 + 1.50 4.15 5.45 5.66 7.29 8.16 9.37 11.08 4.83 (2H, d, NCH ₂ ), 5.11 (2H, m, = CH ₂ ), 5.80 (1H, m, -CH =), 10.66 (1H, s, NH) 1i 1.34 + 1.46 4.15 5.37 5.60 7.26 8.19 9.43 11.04 0.75 + 1.12 (4H, m + m, CH ₂ CH ₂ ), 2.66 (1H, m, NCH), 11.70 + 11.91 (1H, s + s, NH) 1 to 1.44 + 1.50 4.13 5.42 5.67 7.35 10H 8.25 9.42 11.05 11.03 (1H, s, NH) 1l 1.45 + 1.47 4.11 5.40 5.55 7.50 13H 8.12 9.51 11.26 2.17 + 2.19 (6H, s + s, NMe) 1m 1.45 + 1.53 1.62 + 1.66 4.12 5.33 5.71 7.29 8.26 9.11 10.92

Continuation of table 3 1n 1.40 + 1.52 4.15 5.30 5.42 7.23 7.88 8.94 10.61 1.83 (2H, m, CH ₂ ), 2.41 (2H, m, 2CH ₂ ) 1o 1.33 + 1.45 4.13 5.41 5.59 7.42 8.24 9.45 11.10 1.13 (3H, t, Me), 3.51 (4H, m, NCH ₂ + O CH ₂ ), 4.44 (2H, t, OCH ₂ ) 1p 1.41 + 1.50 4.14 5.42 5.62 7.40 8.19 9.36 11.08 1.20-2.45 (10H, m, 5CH ₂ ), 4.37 (1H, NCH) 1p 1.45 + 1.51 4.15 5.42 5.60 6.97 4H
7.43 5H 8.21 9.49 11.03 3.78 (3H, s, OMe) 1s 1.36 + 1.41 4.10 5.34 5.49 7.32 - 8.79 10.94 2.56 (3H, s, = CMe), 3.14 (6H, s, NMe) ∗ Note. For substances 1b, gf, z-k, and r, a doubling of these signals due to the Z / E isomerism relative to the enamine double bond is characteristic

Table 4 Elemental analysis data of target substances No. of substance Found,% Gross formula Calculated,% FROM N N C H N 1a 51.74 4.34 14.37 C21H21N5O7S 51.74 4.34 14.37 1b 52.51 4.60 13.88 C22H23N5O7S 52.69 4.62 13.96 1c 53.65 4.91 13.52 C23H25N5O7S 53.58 4.89 13.58 1g 56.99 5.87 12.13 C27H33N5O7S 56.73 5.82 12.25 1d 58.04 4.78 12.00 C28H27N5O7S 58.22 4.71 12.12 1st 57.44 4.89 11.41 C29H29N5O8S 57.32 4.81 11.53 1g 51.75 4.86 01/13 C23H25N5O6S2 51.97 4.74 13.17 1z 52.86 4.77 12.74 C24H25N5O6S2 53.03 4.64 12.88 1i 52.91 4.73 12.80 C24H25N5O6S2 53.03 4.64 12.88 1 to 55.70 4.44 11.95 C27H25N5O6S2 55.95 4.35 12.08 1l 61.35 4.99 10.12 C35H33N5O6S2 61.48 4.86 10.24 1m 54.68 5.21 8.20 C23H25N3O8S 54.86 5.00 8.35 1n 58.34 5.48 8.66 C23H25N3O6S 58.59 5.34 8.91 1o 53.90 5.57 12.03 C25H29N5O7S2 52.16 5.08 12.17 1p 55.12 5.39 11.74 C27H31N5O6S2 55.37 5.33 11.96 1p 09/09 4.55 11.42 C28H27N5O7S2 55.16 4.46 11.49 1s 54.37 5.18 13.14 C24H27N5O7S 54.43 5.14 13.22

Examples of the study of biological activity

Example 6

Determination of the effectiveness of a substance against gram-positive and gram-negative bacteria.

In the experiments, standard collection strains and bacteria isolated from patients were used. The evaluation was carried out by the method of serial dilutions using nutrient media suitable for the cultivation of the corresponding types of microorganisms.

Compounds were dissolved in sterile water and titrated in concentrations from 500 to 0.025 mg / L. The concentration of the drug in the medium of neighboring tubes was two times different. The result was taken into account after a 72-hour cultivation of bacteria at 37 ° C (table 5).

Table 5 The minimum inhibitory concentration of the obtained compounds Compound MIC, mg / L S. aureus E. coli M. smegmatis Ampicillin
The control 0.05 1.5 12.5 1c 0.03 12.5 12.0 1d 1.5 5.0 12.0 1g 1.0 5.0 12.0 1z 0.25 3.5 6.2 1 to 0.012 1.0 12.0 1l 0.05 3.0 6.2 1o 0.05 5.0 6.2 1p 0.05 5.0 6.2

The results obtained indicate that the claimed compounds are comparable with ampicillin in the level and spectrum of antimicrobial activity, and some surpass the prototype, ampicillin, in certain parameters.

Example 7

Effect on S. aureus in biofilms

Table 6 Survival of S. aureus in biofilms with the claimed compounds (24-hour action) Antibiotic CFU number (Log10) The control 9.9 ± 0.76 Ampicillin 9.78 ± 0.64 1a 8.27 ± 0.34 1b 8.35 ± 0.82 1c 8.32 ± 0.45 1g 8.02 ± 0.16 1d 8.27 ± 0.62 1 to 7.47 ± 0.72 1o 8.18 ± 0.24 1p 8.12 ± 0.72

The data presented indicate that the claimed compounds, in contrast to the prototype ampicillin, are active against staphylococci that are part of biofilms.

Example 8

Effect on S. pyogenes contained in biofilms

Table 7 Survival of S. pyogenes in biofilms with the claimed compounds (24-hour action) Antibiotic CFU number (Log10) The control 8.41 ± 0.76 Ampicillin 8.15 ± 0.66 1c 7.46 ± 0.40 1d 6.44 ± 0.87 1g 7.05 ± 0.15 1z 7.50 ± 0.70 1 to 6.24 ± 0.54 1l 7.20 ± 0.34 1o 8.00 ± 0.65 1p 7.75 ± 0.23

The data presented indicate that the claimed compounds, in contrast to the prototype ampicillin, are active against streptococci that are part of biofilms.

Example 9

Effect on E. coli in biofilms

Table 8 Survival in biofilms under the action of the claimed compounds (24-hour action) Test substance CFU number (Log10) Control (solvent added) 8.97 ± 0.48 Ampicillin 8.64 ± 0.62 1c 7.50 ± 0.15 1d 7.15 ± 0.32 1g 7.27 ± 0.55 1z 7.54 ± 0.34 1 to 7.11 ± 0.65 1l 7.72 ± 0.52

The data presented indicate that the claimed compounds, in contrast to the prototype ampicillin, are active against gram-negative Escherichia, which are part of biofilms.

Example 10

Effect on K. pneumoniae contained in biofilms

Table 9 Survival of K. pneumoniae in biofilms under the action of the claimed compounds (24-hour action) Test substance CFU number (Log10) Control (solvent added) 8.79 ± 0.72 Ampicillin 8.57 ± 0.74 1a 7.49 ± 0.54 1d 7.37 ± 0.14 1g 8.00 ± 0.27 1st 7.78 ± 0.76 1 to 7.02 ± 0.44 1l 7.78 ± 0.76

The data presented indicate that the claimed compounds, in contrast to the prototype ampicillin, are active against gram-negative Klebsiella in biofilms.

Example 11

Effect on A. baumannii contained in biofilms

Table 10 Survival of A. baumannii in biofilms under the action of the claimed compounds (24-hour action) Test substance CFU number (Log10) Control (solvent added) 8.54 ± 0.87 Ampicillin 8.48 ± 0.43 1b 8.11 ± 0.46 1c 7.71 ± 0.15 1d 7.51 ± 0.38 1st 7.79 ± 0.85 1i 7.51 ± 0.75 1 to 6.61 ± 0.55 1o 7.67 ± 0.45 1p 7.41 ± 0.55

The data presented indicate that the claimed compounds, in contrast to the prototype ampicillin, are active against A. baumannii, which are part of biofilms.

Example 12

The penetration of the claimed compounds on S.sonnei inside the cells

To perform the study, HeLa cells were infected with a 24-hour S.sonnei culture previously washed twice by centrifugation from the culture medium and resuspended in phosphate buffer. After a 4-hour exposure, all bacteria that did not enter the cells were killed by gentamicin, which has a bactericidal effect and does not penetrate eukaryotic cells. After exposure with gentamicin, the medium together with the antibiotic (gentamicin) was removed and fresh medium was added with or without test drugs.

Table 11 The effect of the claimed compounds on S.sonnei inside cells N Substance The number of intact cells one. Intact cells 8000 (100%) 2. DMSO 8000 (100%) 3. Infected cells 6000 (75%) four. Ampicillin 6200 (77.5%) 5. 1z 7200 (90%) 6. 1 to 7800 (97.5%) 7. 1l 7500 (93.75%)

The results obtained indicate that the claimed compounds, unlike the prototype, penetrate into human cells and act on the bacteria in them.

The above examples confirm the high antibacterial activity of the substances of the general formula (I), a wide spectrum of action and, very importantly, an effective effect on bacteria that are in human cells and in biofilms.

Claims (1)

Antimicrobial agent with the general formula

where R is selected from the group: H, methyl, ethyl, n-alkyl (C3-C9), phenyl;
X1 and X3 are selected from the group: CH ₂ , O or N (NH or NR, where R is methyl, ethyl, linear or branched alkyl, alkenyl or cycloalkyl C3-C10, CH ₂ CH ₂ OEt, CH ₂ CH ₂ CH ₂ OMe , Ph, a benzene derivative substituted one or more times by linear or branched alkyl, alkenyl or cycloalkyl C1-C9, halogen (F, Cl or Br), CF ₃ , methoxy or another alkoxy, carboxyl or carboxyalkyl, or several of the following substituents CH ₂ Ph, CH ₂ CH ₂ Ph, CH (CH ₃ ) Ph, CH ₂ C ₆ H ₄ (OMeer), CH ₂ C ₆ H ₄ (Fp), CH ₂ CH ₂ C ₆ H ₄ (Fp ) CH ₂ CH ₂ C ₆ H ₃ (3,4-diOMe), 2-furylmethyl, 2- (2-ti nile) ethyl;
X2 - selected from the group: C = O, C = S, CH ₂ , CMe ₂ , C (C ₅ H ₁₀ cyclo).